The subject invention relates to the art of preparing target-associative tags. Such tags are widely used in science, medicine, and elsewhere for a variety of purposes including as aids to visualization or purification. Typically, such tags comprise both a moiety having an ability to associate specifically or preferentially with certain structures [“targeting moiety”] and a moiety having a property or properties useful in discriminating or selecting between members of a set [“discriminating moiety”].
When a target-associative tag associates with its target, a tag-target conjugate is formed. In forming such a tag-target conjugate, the target typically acquires the useful property or properties of the tag. For example, an antibody-associative fluorescent tag can be associated with an antibody to confer the property of fluorescence upon the antibody. The association of the tag and the target in the tag-target conjugate can be based on the formation of one or more covalent bonds, an affinity interaction, a hydrophobic interaction, a hydrogen-bonding interaction, a magnetic interaction, or any other type of interaction that imparts an ability to associate specifically or preferentially with certain structures.
A tag-target conjugate may itself function as a target-associative tag in some cases. For example, when an antibody is tagged with a fluorophore to produce a fluorescent antibody, the fluorescent antibody can then be regarded as target-associative tag that may be directed against structures to which the antibody has an affinity.
There are a wide variety of properties that may be useful in a target-associative tag. In general, any property or properties useful in discriminating or selecting between members of a set could have utility in a target-associative tag. Such properties include, but are not limited to, fluorescence, mass, affinity, reactivity, size, absorbance, magnetism, subatomic spin characteristics, or an ability to associate specifically or preferentially with certain structures.
Practitioners skilled in the art will recognize that although target-associative tags have been described here as often comprising both a targeting moiety and a discriminating moiety, in some cases the classification of a given moiety as a targeting moiety or a discriminating moiety may be ambiguous or subject to context. For instance, a rhodamine moiety incorporated into a target-associative tag might often be regarded as a discriminating moiety in that it may exhibit fluorescence under certain conditions, but the same rhodamine moiety might also be regarded as a targeting moiety in that it may exhibit an affinity interaction with certain structures. In general, the set of all possible targeting moieties is a subset of the set of all possible discriminating moieties; every targeting moiety by definition has a property or properties useful in discriminating or selecting between members of a set in that every targeting moiety is able to specifically or preferentially associate with certain structures. In other words, every targeting moiety is also a discriminating moiety, but there may be discriminating moieties that are not targeting moieties.
While target-associative tags typically comprise both a targeting moiety and a discriminating moiety, it is possible that a single moiety could fulfill both roles. For example, in certain fluorogenic reagents known in the art it might be difficult, impossible, or simply of little descriptive utility to identify separate targeting and discriminating moieties. For example, 7-N,N-dimethylsulfonyl-4-(2,1,3-benzoxadiazolyl)isothiocyanate, also known as DBD-NCS, is a fluorogenic reagent used in peptide sequencing analysis. In associating with its target via the formation of a covalent bond, DBD-NCS is converted from a fluorogenic form to a fluorescent form. Thus the target-reactive moiety is a component of the fluorogenic/fluorescent moiety. Also, just as it is possible that a single moiety could fulfill the roles of both discriminating moiety and targeting moiety, it is also possible that a target-associative tag could contain multiple discriminating moieties or multiple targeting moieties or both.
One way of discriminating between members of a set is by fluorescent emission. For instance, the set of all structures within a cell can be differentiated by degree of fluorescence when visualizing the cell. Fluorescent target-associative tags are widely used for this purpose. For example, phalloidin is a cyclic peptide produced by the poisonous mushroom, Amanita phalloides. Conjugation of phalloidin as the targeting moiety and the fluorophore rhodamine as the discriminating moiety produces a target-associative tag that associates preferentially with actin bundles, allowing visualization of said actin bundles by fluorescence microscopy. Another common type of fluorescent target-associative tag is formed by conjugation of a fluorophore and an antibody. Such a fluorescent target-associative tag can be directed against structures to which the antibody has an affinity. For example, a target-associative tag consisting of the fluorophore Texas Red conjugated with a goat anti-mouse immunoglobulin can be directed against mouse primary antibodies for the purpose of visualizing cell structures.
Historically, initial difficulties in establishing the localization of phosphorylated residues in proteins led to the development of a scheme by which phosphoserine and phosphothreonine residues were modified by beta-elimination followed by nucleophilic attack to give a derivatized residue. Such a scheme has been known in the art for at least 32 years. [Simpson, D. L. et al., Biochemistry 111:1849-1856, 1972; Kolesnikova, V. Y. et al., Biokhimiya 39:235-240 (Engl. Trans.) 1974]. Subsequent refinement of the technique has frequently favored thiols as the preferred nucleophile, and the use of thiols as nucleophiles in the scheme has been known in the art for at least 25 years. [Clark, R. C. and Dijkstra, J., Int. J. Biochem. 11:577-585, 1979].
Many modifications of the beta-elimination/nucleophilic attack technique are known in the art, and such modifications frequently involve the use of target-associative tags as nucleophiles to imbue the derivatized phosphoresidues with a desired property. One type of modification incorporates fluorophores. Use of pyridoxamine and fluorescence detection allows detection of low picomolar quantities of a phosphorylated polypeptide. [Hastings, T. G. and Reimann E. M, FEB 231(2):431-436, 1988]. Use of fluorescein and laser-induced fluorescence allows detection of attomolar quantities of phosphoserine-containing peptides and proteins. [Fadden, P. and Haystead, T. A., Anal. Biochem. 225(1):81-88, 1995].
Another type of modification incorporates an affinity tag. Beta-elimination and subsequent nucleophilic attack by ethanedithiol, followed by addition of biotin to the resulting free thiol group, allows for affinity isolation and enrichment of protein fragments containing phosphorylated residues. [Adamczyk, M. et al., Rapid Comm. Mass. Spec. 15:1481-1488, 2001; Oda, Y. et al., Nature Biotechnol. 19:379-382, 2001]. A further refinement that utilizes nucleophilic attack by a tag containing both biotin and isotopic mass markers may be particularly useful for mass spectroscopy following affinity purification. [Goshe, M. B. et al., Anal. Chem. 73:2578-2586, 2001; Goshe, M. B. et al., Anal. Chem. 74:607-616, 2001]. An introduced thiol tag can also be used directly for affinity purification on an activated thiol resin. [McLachlin, D. T. and Chait, B. T., Anal. Chem. 75(24):6826-6836, 2003].
Target-associative tags that mimic lysine have been used in the beta-elimination/Michael addition scheme to introduce additional enzyme-mediated proteolysis sites at phosphoserine and phosphothreonine residues, thus facilitating the analysis of proteins containing such residues. [Knight, Z. A. et al., Nature Biotechnol. 21(9):1047-1054, 2003].